Développement des marqueurs moléculaires spécifiques pour l'identification et la quantification de deux espèces de champignons mycorhiziens arbusculaires en utilisant la PCR en temps réel

Les champignons mycorhizien à arbuscules (CMA) sont des organismes pouvant établir des symbioses avec 80% des plantes terrestres. Les avantages d'une telle symbiose sont de plus en plus caractérisés et exploités en agriculture. Par contre, jusqu'à maintenant, il n'existe aucun outil permettant à la fois l'identification et la quantification de ces champignons dans le sol de façon fiable et rapide. Un tel outil permettrait, entre autres, de mieux comprendre les dynamiques des populations des endomycorhizes dans le sol. Pour les producteurs d'inoculum mycorhiziens, cela permettrait également d'établir un suivi de leurs produits en champs et d'avoir un contrôle de qualité de plus sur leurs inoculants. C'est ce que nous avons tenté de développer au sein du laboratoire du Dr. Hijri. Depuis environ une trentaine d'années, des outils d'identification et/ou de quantification ont été développés en utilisant les profiles d'acides gras, les isozymes, les anticorps et finalement l'ADN nucléaire. À ce jour, ces méthodes d’identification et de quantification sont soit coûteuses, soit imprécises. Qui plus est, aucune méthode ne permet à la fois la quantification et l’identification de souches particulières de CMA. L’ADN mitochondrial ne présente pas le même polymorphisme de séquence que celui qui rend l’ADN nucléaire impropre à la quantification. C'est pourquoi nous avons analysé les séquences d’ADN mitochondrial et sélectionné les régions caractéristiques de deux espèces de champignons mycorhiziens arbusculaires (CMA). C’est à partir de ces régions que nous avons développé des marqueurs moléculaires sous forme de sondes et d’amorces TaqMan permettant de quantifier le nombre de mitochondries de chacune de ces espèces dans un échantillon d’ADN. Nous avons ensuite tenté de déterminer une unité de quantification des CMA, soit un nombre de mitochondries par spore. C’est alors que nous avons réalisé que la méthode de préparation des échantillons de spores ainsi que la méthode d’extraction d’ADN avaient des effets significatifs sur l’unité de quantification de base. Nous avons donc optimisé ces protocoles, avant d’en e tester l’application sur des échantillons de sol et de racines ayant été inoculés avec chacune des deux espèces cibles. À ce stade, cet outil est toujours semi-quantificatif, mais il permet 9 l’identification précise de deux espèces de CMA compétentes dans des milieux saturés en phosphore inorganique. Ces résultats , en plus d’être prometteurs, ont permis d’augmenter les connaissances méthodologiques reliées à la quantification des CMA dans le sol, et suggèrent qu’à cause de leurs morphologies différentes, l’élaboration d’un protocole de quantification standardisé pour toutes les espèces de CMA demeure un objectif complexe, qui demande de nouvelles études in vivo.

Assessment of PEG on Polymeric Particles Surface, a Key Step in Drug Carrier Translation

Injectable drug nanocarriers have greatly benefited in their clinical development from the addition of a superficial hydrophilic corona to improve their cargo pharmacokinetics. The most studied and used polymer for this purpose is poly(ethylene glycol), PEG. However, in spite of its wide use for over two decades now, there is no general consensus on the optimum PEG chain coverage-density and size required to escape from the mononuclear phagocyte system and to extend the circulation time. Moreover, cellular uptake and active targeting may have conflicting requirements in terms of surface properties of the nanocarriers which complicates even more the optimization process. These persistent issues can be largely attributed to the lack of straightforward characterization techniques to assess the coverage-density, the conformation or the thickness of a PEG layer grafted or adsorbed on a particulate drug carrier and is certainly one of the main reasons why so few clinical applications involving PEG coated particle-based drug delivery systems are under clinical trial so far. The objective of this review is to provide the reader with a brief description of the most relevant techniques used to assess qualitatively or quantitatively PEG chain coverage-density, conformation and layer thickness on polymeric nanoparticles. Emphasis has been made on polymeric particle (solid core) either made of copolymers containing PEG chains or modified after particle formation. Advantages and limitations of each technique are presented as well as methods to calculate PEG coverage-density and to investigate PEG chains conformation on the NP surface.

Polyphosphate Accumulation by Marine Bacteria

Phosphate (Pi) is one among the most important essential residues in maintenance and inheritance of life, with far diverse physiological role as structural, functional and energy transduction. Phosphate accumulation in wastewaters containing run off of fertilizers and industrial discharges is a global problem that results in algal blooms in bays, lakes and waterways. Currently available methods for removing phosphates from wastewater are based primarily on polyP accumulation by the activated sludge bacteria. PolyP plays a critical role in several environmental and biotechnological problems. Possible relation of interaction between polyP accumulation phenomenon, the low biomass, low Pi uptake, and varying results obtained in response to the impact of sodium chloride, pH, temperature, various inorganic salts and additional carbon sources studied, are all intriguing observations in the present investigation. The results of the present study have evidenced very clearly the scope for potential strains of bacteria from both sea water and marine sediments which could be exploited both for Pi removal in wastewater released by industries and intensive aquaculture practices in to the aquatic environment as well as to harness the potential strains for industrial production of polyP which was wide range of applications.

Studies on Larval Nutrition in The Pearl Oyster Pinctada Pucata (Gould)

The present study has yielded a great deal of information on nutrition of pearl oyster larvae. T he formulae presented may be used effectively and with advantage in improving the larval rearing system with specific reference to nutritional aspects. It is also hoped that this is the first comprehensive study on pearl oyster larval nutrition would stimulate further detailed investigations on many of the other finer aspects of tropical bivalve larval nutrition.

Studies on arbuscular mycorrhiza (AM) in the Alentejo (Portugal) using pea mutants resistant to AM fungi as a control tool for field conditions

The utilization and management of arbuscular mycorrhiza (AM) symbiosis may improve production and sustainability of the cropping system. For this purpose, native AM fungi (AMF) were sought and tested for their efficiency to increase plant growth by enhanced P uptake and by alleviation of drought stress. Pot experiments with safflower (Carthamus tinctorius) and pea (Pisum sativum) in five soils (mostly sandy loamy Luvisols) and field experiments with peas were carried out during three years at four different sites. Host plants were grown in heated soils inoculated with AMF or the respective heat sterilized inoculum. In the case of peas, mutants resistant to AMF colonization were used as non-mycorrhizal controls. The mycorrhizal impact on yields and its components, transpiration, and P and N uptake was studied in several experiments, partly under varying P and N levels and water supply. Screening of native AMF by most probable number bioassays was not very meaningful. Soil monoliths were placed in the open to simulate field conditions. Inoculation with a native AMF mix improved grain yield, shoot and leaf growth variables as compared to control. Exposed to drought, higher soil water depletion of mycorrhizal plants resulted in a haying-off effect. The growth response to this inoculum could not be significantly reproduced in a subsequent open air pot experiment at two levels of irrigation and P fertilization, however, safflower grew better at higher P and water supply by multiples. The water use efficiency concerning biomass was improved by the AMF inoculum in the two experiments. Transpiration rates were not significantly affected by AM but as a tendency were higher in non-mycorrhizal safflower. A fundamental methodological problem in mycorrhiza field research is providing an appropriate (negative) control for the experimental factor arbuscular mycorrhiza. Soil sterilization or fungicide treatment have undesirable side effects in field and greenhouse settings. Furthermore, artificial rooting, temperature and light conditions in pot experiments may interfere with the interpretation of mycorrhiza effects. Therefore, the myc- pea mutant P2 was tested as a non-mycorrhizal control in a bioassay to evaluate AMF under field conditions in comparison to the symbiotic isogenetic wild type of var. FRISSON as a new integrative approach. However, mutant P2 is also of nod- phenotype and therefore unable to fix N2. A 3-factorial experiment was carried out in a climate chamber at high NPK fertilization to examine the two isolines under non-symbiotic and symbiotic conditions. P2 achieved the same (or higher) biomass as wild type both under good and poor water supply. However, inoculation with the AMF Glomus manihot did not improve plant growth. Differences of grain and straw yields in field trials were large (up to 80 per cent) between those isogenetic pea lines mainly due to higher P uptake under P and water limited conditions. The lacking N2 fixation in mutants was compensated for by high mineral N supply as indicated by the high N status of the pea mutant plants. This finding was corroborated by the results of a major field experiment at three sites with two levels of N fertilization. The higher N rate did not affect grain or straw yields of the non-fixing mutants. Very efficient AMF were detected in a Ferric Luvisol on pasture land as revealed by yield levels of the evaluation crop and by functional vital staining of highly colonized roots. Generally, levels of grain yield were low, at between 40 and 980 kg ha-1. An additional pot trial was carried out to elucidate the strong mycorrhizal effect in the Ferric Luvisol. A triplication of the plant equivalent field P fertilization was necessary to compensate for the mycorrhizal benefit which was with five times higher grain yield very similar to that found in the field experiment. However, the yield differences between the two isolines were not always plausible as the evaluation variable because they were also found in (small) field test trials with apparently sufficient P and N supply and in a soil of almost no AMF potential. This similarly occurred for pea lines of var. SPARKLE and its non-fixing mycorrhizal (E135) and non-symbiotic (R25) isomutants, which were tested in order to exclude experimentally undesirable benefits by N2 fixation. In contrast to var. FRISSON, SPARKLE was not a suitable variety for Mediterranean field conditions. This raises suspicion putative genetic defects other than symbiotic ones may be effective under field conditions, which would conflict with the concept of an appropriate control. It was concluded that AMF resistant plants may help to overcome fundamental problems of present research on arbuscular mycorrhiza, but may create new ones.

Efecto de la hipoxia-reoxigenación y las radiaciones ionizantes en la captación de glucosa en líneas tumorales de seno y colon cocultivadas con células endoteliales.

La captación de glucosa y su conversión en lactato juega un papel fundamental en el metabolismo tumoral, independientemente de la concentración de oxígeno presente en el tejido (efecto Warburg). Sin embrago, dicha captación varía de un tipo tumoral a otro, y dentro del mismo tumor, situación que podría depender de las características microambientales tumorales (fluctuaciones de oxígeno, presencia de otros tipos celulares) y de factores estresores asociados a los tratamientos. Se estudió el efecto de la hipoxia-reoxigenación (HR) y las radiaciones ionizantes (RI) sobre la captación de glucosa, en cultivos de líneas tumorales MCF-7 y HT-29, cultivadas de forma aislada o en cocultivo con la línea celular EAhy296. Se encontró que la captación de glucosa en HR es diferente para lo descrito en condiciones de hipoxia permanente y que es modificada en el cocultivo. Se identificaron poblaciones celulares dentro de la misma línea celular, de alta y baja captación de glucosa, lo que implicaría una simbiosis metabólica de la célula como respuesta adaptativa a las condiciones tumorales. Se evaluó la expresión de NRF2 y la translocación nuclear de NRF2 y HIF1a, como vías de respuesta a estrés celular e hipoxia. La translocación nuclear de las proteínas evaluadas explicaría el comportamiento metabólico de las células tumorales de seno, pero no de colon, por lo cual deben existir otras vías metabólicas implicadas. Las diferencias en el comportamiento de las células tumorales en HR en relación con hipoxia permitirá realizar planeaciones dosimétricas más dinámicas, que reevalúen las condiciones de oxigenación tumoral constantemente.

[177Lu – DOTA – Tyr3] – OCTREOTATE para el tratamiento de tumores neuroendocrinos gastroenteropancreáticos. Revisión sistemática de la literatura.

Introducción: los tumores neuroendocrinos gastroenteropancreáticos se diagnostican en estadio avanzado en 60 - 80% de los pacientes y las opciones terapéuticas son limitadas. Se realizó una revisión sobre el beneficio clínico del tratamiento con [177Lu - DOTA - Tyr3] - Octreotate en pacientes con enfermedad metastásica o inoperable. Objetivos: evaluar la eficacia, impacto en calidad de vida y toxicidad de la terapia con 177Lu DOTATE en pacientes con tumores neuroendocrinos gastroenteropancreáticos avanzados. Materiales y Métodos: se condujo una revisión sistemática de la literatura mediante la búsqueda de estudios clínicos prospectivos y retrospectivos en bases electrónicas (MEDLINE, EMBASE, LILACS, SCIELO, OVID y la Biblioteca Cochrane) de cualquier idioma, año y estado de publicación. Se incluyeron 5 estudios, por la heterogeneidad existente entre los estudios no se realizó un metaanálisis. Resultados: la respuesta tumoral global fue del 45 - 57%, la enfermedad permaneció estable en 27% - 38% y progresó en 6% - 21% de casos en las series incluidas. El tiempo libre de progresión osciló entre 31 - 40 meses y la sobrevida global de 31– 51 meses. Se observó toxicidad hematológica grado 3-4 hasta en 9.5% de pacientes. Hubo mejoría significativa en la calidad de vida de pacientes tratados con 177LuDOTATATE. Conclusiones: la terapia con 177Lu- DOTATATE ofrece un beneficio clínico a los pacientes con tumores neuroendocrinos bien diferenciados avanzados por su impacto positivo en calidad de vida, control de síntomas, ralentiza la progresión tumoral y su toxicidad es baja.

La génesis de las parafilias sexuales y la homosexualidad egodistónica: el Modelo de los Mecanismos Tensionales

Mediante el desarrollo del Modelo de los Mecanismos Tensionales, basado en el trastorno de evitación experiencial (Hayes, Wilson, Gifford, Follette & Stroshal,1996) y en la Teoría de la Terminación Conductual propuesta por McConaghy (1980), el presente artículotrata de exponer una serie de fenómenos que pueden estar pasando desapercibidos a la hora de explicar la génesis y el mantenimiento de las parafilias sexuales y la homosexualidad egodistónica. Para ello se parte del análisis de los testimonios de sujetos parafílicos y homosexuales egodistónicos (que no aceptan su homosexualidad) registrados durante los años 2005 y 2006. Desde una perspectiva de la psicología contextual y haciendo uso del análisis funcional y topográfico de las conductas características de cada trastorno, se observará cómo ambas manifestaciones sexuales tienen aspectos comunes (factores predisponentes, precipitantes y demantenimiento) en su origen y evolución.

Bioquímica en la vida diaria: el equilibrio químico y la función transportadora de la hemoglobina

La hemoglobina es una proteína sanguínea que puede transportar oxígeno, un gas insoluble en medio acuoso, llevándolo a las diferentes partes del organismo en donde es requerido para su buen funcionamiento, así como productos metabólicos como el CO2 y el hidrógeno, para su excreción. Estos procesos se ven condicionados por factores como el pH, la concentración de BPG, las presiones parciales de O2 y CO2, la cooperatividad de la unión entre la hemoglobina y esos compuestos y los cambios conformacionales que la hemoglobina debe sufrir para captar y soltar eficientemente estas moléculas en el sitio del organismo donde son requeridos. Cambios abruptos en la presión atmosférica ligados a la altura, y la exposición a altas concentraciones de otros gases afines a la hemoglobina como el monóxido de carbono, presente en vehículos o recintos cerrados, pueden comprometer el funcionamiento normal del organismo precisamente porque causan efectos sobre esa función transportadora de la hemoglobina. En este escrito, se explicarán fenómenos de la vida diaria relacionados con el transporte de gases por la hemoglobina, como una demostración de que los conocimientos bioquímicos comienzan a ser útiles desde ahora para entender situaciones cotidianas y a dejarnos la expectativa de su valor para entender muchos de los problemas de salud que tendremos en nuestras manos.

Impacto de un programa de promoción de actividad física en mujeres trabajadoras de dos empresas de Bogotá

Estilos de vida activos y saludables se han asociado con mejoras en calidad de vida de mujeres trabajadoras. Objetivos: establecer la efectividad de un programa de actividad física para mejorar componentes del fitness en mujeres trabajadoras de dos empresas de Bogotá. Materiales y métodos: se realizó un estudio longitudinal, descriptivo de carácter exploratorio, con mediciones previas y posteriores de variables cuantitativas de componentes del fitness. Se incluyeron mujeres trabajadorasde 24 a 49 años, sin factores de riesgos asociados y sin restricciones para la práctica de actividad física de acuerdo con el cuestionario PARQ & YOU. Quince mujeres (37,33 ±9,3) se sometieron voluntariamente a la intervención en actividad física durante un período de 12 semanas, 60 minutos diarios. Para el análisis estadístico de los datos, se empleó el paquete estadístico SPSS 17. Resultados: se presentaron cambios significativos después de la intervención en el índice de masa corporal (IMC) (p = 0,023), resistencia abdominal (p = 0,004) y fuerza en miembros inferiores (MMII) (p = 0,001). No hubo cambios estadísticamente significativos en consumo pico de oxígeno y flexibilidad. Conclusión: a partir de estos resultados, se pudo establecer que el protocolo del programa de promoción de actividad física aplicado produjo cambios en el fitness de las mujeres que lo completaron, lo que significa que este puede ser replicado con resultados efectivos tras su desarrollo.

An application of the GLUE methodology for estimating the parameters of the INCA-N model

The conceptual and parameter uncertainty of the semi-distributed INCA-N (Integrated Nutrients in Catchments-Nitrogen) model was studied using the GLUE (Generalized Likelihood Uncertainty Estimation) methodology combined with quantitative experimental knowledge, the concept known as 'soft data'. Cumulative inorganic N leaching, annual plant N uptake and annual mineralization proved to be useful soft data to constrain the parameter space. The INCA-N model was able to simulate the seasonal and inter-annual variations in the stream-water nitrate concentrations, although the lowest concentrations during the growing season were not reproduced. This suggested that there were some retention processes or losses either in peatland/wetland areas or in the river which were not included in the INCA-N model. The results of the study suggested that soft data was a way to reduce parameter equifinality, and that the calibration and testing of distributed hydrological and nutrient leaching models should be based both on runoff and/or nutrient concentration data and the qualitative knowledge of experimentalist. (c) 2006 Elsevier B.V. All rights reserved.

Eutrophication control in river-systems: an application of INCA-P to the River Lugg

The Integrated Catchments model of Phosphorus dynamics (INCA-P) was applied to the River Lugg to determine the key factors controlling delivery of phosphorus to the main channel and to quantify the relative contribution of diffuse and point sources to the in-stream phosphorus (P) load under varying hydrological conditions. The model is able to simulate the seasonal variations and inter-annual variations in the in-stream total-phosphorus concentrations. However, difficulties in simulating diffuse inputs arise due to equifinality in the model structure and parameters. The River Lugg is split into upper and lower reaches. The upper reaches are dominated by grassland and woodland, so the patterns in the stream-water total-phosphorus concentrations are typical of diffuse source inputs; application of the model leads to estimates of the relative contribution to the in-stream P load from diffuse and point sources as 9:1. In the lower reaches, which are more intensively cultivated and urbanised, the stream-water total-phosphorus concentration dynamics are influenced more by point-sources; the simulated relative diffuse/point contribution to the in-stream P load is 1: 1. The model set-up and simulations are used to identify the key source-areas of P in the catchment, the P contribution of the Lugg to the River Wye during years with contrasting precipitation inputs, and the uptake and release of P from within-reach sediment. In addition, model scenarios are run to identify the impacts of likely P reductions at sewage treatment works on the in-stream soluble-reactive P concentrations and the suitability of this as a management option is assessed for reducing eutrophication.

Modelling the inorganic nitrogen behaviour in a small Mediterranean forested catchment, Fuirosos (Catalonia)

The aim of this work was to couple a nitrogen (N) sub-model to already existent hydrological lumped (LU4-N) and semi-distributed (LU4-R-N and SD4-R-N) conceptual models, to improve our understanding of the factors and processes controlling nitrogen cycling and losses in Mediterranean catchments. The N model adopted provides a simplified conceptualization of the soil nitrogen cycle considering mineralization, nitrification, immobilization, denitrification, plant uptake, and ammonium adsorption/desorption. It also includes nitrification and denitrification in the shallow perched aquifer. We included a soil moisture threshold for all the considered soil biological processes. The results suggested that all the nitrogen processes were highly influenced by the rain episodes and that soil microbial processes occurred in pulses stimulated by soil moisture increasing after rain. Our simulation highlighted the riparian zone as a possible source of nitrate, especially after the summer drought period, but it can also act as an important sink of nitrate due to denitrification, in particular during the wettest period of the year. The riparian zone was a key element to simulate the catchment nitrate behaviour. The lumped LU4-N model (which does not include the riparian zone) could not be validated, while both the semi-distributed LU4-R-N and SD4-R-N model (which include the riparian zone) gave satisfactory results for the calibration process and acceptable results for the temporal validation process.

Exploring options for managing strategies for pea-barley intercropping using a modeling approach

A modeling Study was carried out into pea-barley intercropping in northern Europe. The two objectives were (a) to compare pea-barley intercropping to sole cropping in terms of grain and nitrogen yield amounts and stability, and (b) to explore options for managing pea-barley intercropping systems in order to maximize the biomass produced and the grain and nitrogen yields according to the available resources, such as light, water and nitrogen. The study consisted of simulations taking into account soil and weather variability among three sites located in northern European Countries (Denmark, United Kingdom and France), and using 10 years of weather records. A preliminary stage evaluated the STICS intercrop model's ability to predict grain and nitrogen yields of the two species, using a 2-year dataset from trials conducted at the three sites. The work was carried out in two phases, (a) the model was run to investigate the potentialities of intercrops as compared to sole crops, and (b) the model was run to explore options for managing pea-barley intercropping, asking the following three questions: (i) in order to increase light capture, Would it be worth delaying the sowing dates of one species? (ii) How to manage sowing density and seed proportion of each species in the intercrop to improve total grain yield and N use efficiency? (iii) How to optimize the use of nitrogen resources by choosing the most suitable preceding crop and/or the most appropriate soil? It was found that (1) intercropping made better use of environmental resources as regards yield amount and stability than sole cropping, with a noticeable site effect, (2) pea growth in intercrops was strongly linked to soil moisture, and barley yield was determined by nitrogen uptake and light interception due to its height relative to pea, (3) sowing barley before pea led to a relative grain yield reduction averaged over all three sites, but sowing strategy must be adapted to the location, being dependent on temperature and thus latitude, (4) density and species proportions had a small effect on total grain yield, underlining the interspecific offset in the use of environmental growth resources which led to similar total grain yields whatever the pea-barley design, and (5) long-term strategies including mineralization management through organic residue supply and rotation management were very valuable, always favoring intercrop total grain yield and N accumulation. (C) 2009 Elsevier B.V. All rights reserved.

Nitrogen budgets for three cropping systems fertilised with cattle manure

A field plot experiment was set up on a sandy loam soil of SW England in order to determine the efficiency of nitrogen use from different cattle manures. The manure treatments were low and high dry matter cattle slurries and one farmyard manure applied at a target rate of 200 kg total Nha(-1) year(-1), and an untreated control. There were three different cropping systems: ryegrass/clover mixture, maize/rye and maize/bare soil, which were evaluated during 1998/99 and 1999/00. Measurements were made of N losses, N uptake and herbage DM yields. Result showed that manure type had a significant effect on N utilisation only for maize. N balances were negative in maize (approximately -247 to -10 kg N) compared to grass (approximately 5-158 kg N). Agronomic management was more important than manure type in influencing N losses, where soil cultivation appeared to be a key factor when comparing maize and grass systems. (C) 2004 Elsevier Ltd. All rights reserved.